Post-relapse treatment of CD30 expressing lymphomas

ABSTRACT

Methods for the retreatment of CD30-expressing lymphomas with an anti-CD30 vc-PAB-MMAE antibody-drug conjugate are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/293,311 filed Jan. 8, 2010, the disclosure of which is incorporatedby reference herein.

FIELD

The present invention relates, inter alia, to methods for thepost-relapse treatment of a subject having a previously treated CD30expressing lymphoma.

BACKGROUND

Hodgkin lymphoma (HL) is a neoplasm of lymphoid tissue that is definedhistopathologically by the presence of the malignantHodgkin-Reed-Sternberg (HRS) cells. The characteristic surface antigenexpressed on HRS cells is CD30. There are an estimated 8,000 new HLcases diagnosed annually in the United States and Canada. Advances inthe use of combined chemotherapy and radiotherapy in HL over the pasthalf-century have resulted in a durable remission rate of approximately70%. However, these multi-agent regimens confer a significant morbidityon patients, including secondary malignancies, cardiac disease, andinfertility. Furthermore, approximately 30% of patients presenting withHL will become refractory to initial therapy or will relapse. Salvagechemotherapy regimens and autologous stem cell transplant (ASCT) aresecondary options for these patients, but both are associated withsignificant morbidity and limited long term disease control. Patientswho relapse after ASCT or are ineligible for salvage therapy have a verypoor prognosis. Currently, there is a lack of well-tolerated,efficacious treatment options for these patients. Thus, there continuesto be an unmet medical need for patients suffering from HL and otherCD30 expressing lymphomas. The present invention addresses this andother needs.

DESCRIPTION OF EXEMPLARY EMBODIMENTS A. General Introduction

The present invention provides, inter alia, methods for the post-relapsetreatment of a subject having a previously treated CD30-expressinglymphoma. The present inventors have discovered that a subject that hadpreviously been treated with a CD30 targeted therapy, discontinuedtreatment after achieving significant benefit, and subsequentlyrelapsed, can still achieve tumor regression if treated with ananti-CD30 vc-PAB-MMAE antibody-drug conjugate.

The CD30 antigen is expressed in large numbers on tumor cells of selectlymphomas. Hodgkin lymphoma and anaplastic large-cell lymphoma areexamples of CD30-expressing lymphomas that can be treated by the presentmethods. In an embodiment of the present invention, the subject to betreated with the present methods has a relapsed CD30 expressinglymphoma. In one embodiment, the relapsed CD30 expressing lymphoma isHodgkin lymphoma. In another embodiment the relapsed CD30 expressinglymphoma is anaplastic large-cell lymphoma.

The subjects to be treated with the methods of the present invention arethose that (i) have been previously diagnosed as having a CD30expressing lymphoma, (ii) have been previously treated with a CD30targeted therapy, e.g., an antibody or antibody-drug conjugate directedagainst the CD30 antigen, (iii) achieved clinical benefit as a result ofthe CD30 targeted therapy (e.g., the subject had stable disease withdecreasing tumor volume, a partial response or a complete response asdetermined by guidelines set forth in Cheson et al., Journal of ClinicalOncology, 25(5), 2007, 579-586, incorporated herein by reference in itsentirety and for all purposes), (iv) discontinued treatment with theCD30 targeted therapy after achieving clinical benefit, and (v)subsequently relapsed. In some embodiments, following treatment with theCD30 targeted therapy, the subject underwent further treatment regimensincluding chemotherapy and/or a stem cell transplant. In someembodiments, the time between discontinuation of previous treatment withthe CD30 targeted therapy and treatment with the present methods is 10years or less, 8 years or less, 5 years or less, or 3 years or less.

B. Definitions and Abbreviations

“Cytotoxic effect,” in reference to the effect of an agent on a cell,means killing of the cell. “Cytostatic effect” means an inhibition ofcell proliferation. A “cytotoxic agent” means an agent that has acytotoxic or cytostatic effect on a cell, thereby depleting orinhibiting the growth of, respectively, cells within a cell population.

As used herein, the terms “treatment” or “treat” refer to slowing,stopping, or reversing the progression of a CD30-expressing lymphoma ina subject.

The terms “specific binding” and “specifically binds” mean that theanti-CD30 antibody will react, in a highly selective manner, with itscorresponding target, CD30 and not with the multitude of other antigens.Typically, the anti-CD30 antibody binds with an affinity of at leastabout 1×10⁻⁷ M, and preferably 1×10⁻⁸ M to 1×10⁻⁹ M, 1×10⁻¹⁰ M, 1×10⁻¹¹M, or 1×10⁻¹² M.

The terms “antibody” is used herein in the broadest sense andspecifically encompass full-length and native antibodies, monoclonalantibodies (including full-length monoclonal antibodies), polyclonalantibodies, multispecific antibodies (e.g., bispecific antibodies), andantibody fragments thereof, such as variable domains and other portionsof antibodies that exhibit a desired biological activity. Antibodyfragments for use herein retain their ability to specifically bind tothe CD30 antigen

The term “monoclonal antibody” refers to an antibody that is derivedfrom a single cell clone, including any eukaryotic or prokaryotic cellclone, or a phage clone, and not the method by which it is produced.Thus, the term “monoclonal antibody” as used herein is not limited toantibodies produced through hybridoma technology.

The terms “identical” or “percent identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of nucleotides or amino acid residues that are the same, whencompared and aligned for maximum correspondence. To determine thepercent identity, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in the sequence of a first aminoacid or nucleic acid sequence for optimal alignment with a second aminoor nucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=# ofidentical positions/total # of positions (e.g., overlappingpositions)×100). In certain embodiments, the two sequences are the samelength.

The term “substantially identical,” in the context of two nucleic acidsor polypeptides, refers to two or more sequences or subsequences thathave at least 70% or at least 75% identity; more typically at least 80%or at least 85% identity; and even more typically at least 90%, at least95%, or at least 98% identity (as determined using one of the methodsset forth).

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. A preferred, non-limitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl.Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul, 1993,Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul, et al.,1990, J. Mol. Biol. 215:403-410. BLAST nucleotide searches can beperformed with the NBLAST program, score=100, wordlength=12 to obtainnucleotide sequences homologous to a nucleic acid encoding a protein ofinterest. BLAST protein searches can be performed with the XBLASTprogram, score=50, wordlength=3 to obtain amino acid sequenceshomologous to protein of interest. To obtain gapped alignments forcomparison purposes, Gapped BLAST can be utilized as described inAltschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively,PSI-Blast can be used to perform an iterated search which detectsdistant relationships between molecules (Id.). When utilizing BLAST,Gapped BLAST, and PSI-Blast programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used. Anotherpreferred, non-limiting example of a mathematical algorithm utilized forthe comparison of sequences is the algorithm of Myers and Miller, CABIOS(1989). Such an algorithm is incorporated into the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used. Additional algorithms for sequenceanalysis are known in the art and include ADVANCE and ADAM as describedin Torellis and Robotti, 1994, Comput. Appl. Biosci. 10:3-5; and FASTAdescribed in Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. 85:2444-8.Within FASTA, ktup is a control option that sets the sensitivity andspeed of the search. If ktup=2, similar regions in the two sequencesbeing compared are found by looking at pairs of aligned residues; ifktup=1, single aligned amino acids are examined. ktup can be set to 2 or1 for protein sequences, or from 1 to 6 for DNA sequences. The defaultif ktup is not specified is 2 for proteins and 6 for DNA.

Alternatively, protein sequence alignment may be carried out using theCLUSTAL W algorithm, as described by Higgins et al., 1996, MethodsEnzymol. 266:383-402.

The term “pharmaceutically acceptable” as used herein refers to thosecompounds, materials, compositions, and/or dosage forms that are, withinthe scope of sound medical judgment, suitable for contact with thetissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problems or complicationscommensurate with a reasonable benefit/risk ratio. The term“pharmaceutically compatible ingredient” refers to a pharmaceuticallyacceptable diluent, adjuvant, excipient, or vehicle with which anantibody-drug conjugate is administered.

The phrase “CD30 targeted therapy” refers to therapy targeted to theCD30 antigen. CD30 targeted therapy includes unconjugated anti-CD30antibodies and anti-CD30 antibodies conjugated to cytotoxic drugs. Insome embodiments, the CD30 targeted therapy is therapy with an anti-CD30vc-PAB-MMAE antibody-drug conjugate.

The abbreviation “MMAE” refers to monomethyl auristatin E.

The abbreviations “vc” and “val-cit” refer to the dipeptidevaline-citrulline.

The abbreviation “PAB” refers to the self-immolative spacer:

The abbreviation “MC” refers to the stretcher maleimidocaproyl:

cAC10-MC-vc-PAB-MMAE refers to a chimeric AC10 antibody conjugated tothe drug MMAE through a MC-vc-PAB linker.

An anti-CD30 vc-PAB-MMAE antibody-drug conjugate refers to an anti-CD30antibody conjugated to the drug MMAE via a linker comprising thedipeptide valine citrulline and the self-immolative spacer PAB as shownabove.

As used herein, “pharmaceutically acceptable salts” refer to derivativesof the disclosed compounds wherein the parent compound is modified bymaking acid or base salts thereof. Examples of pharmaceuticallyacceptable salts include, but are not limited to, mineral or organicacid salts of basic residues such as amines; alkali or organic salts ofacidic residues such as carboxylic acids; and the like. Thepharmaceutically acceptable salts include the conventional non-toxicsalts or the quaternary ammonium salts of the parent compound formed,for example, from non-toxic inorganic or organic acids. For example,such conventional non-toxic salts include those derived from inorganicacids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric,nitric and the like; and the salts prepared from organic acids such asacetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric,citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic,benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic,and the like. These physiologically acceptable salts are prepared bymethods known in the art, e.g., by dissolving the free amine bases withan excess of the acid in aqueous alcohol, or neutralizing a freecarboxylic acid with an alkali metal base such as a hydroxide, or withan amine.

Unless otherwise noted, the term “alkyl” refers to a saturated straightor branched hydrocarbon having from about 1 to about 20 carbon atoms(and all combinations and subcombinations of ranges and specific numbersof carbon atoms therein), with from about 1 to about 8 carbon atomsbeing preferred. Examples of alkyl groups are methyl, ethyl, n-propyl,iso-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, n-pentyl,2-pentyl, 3-pentyl, 2-methyl-2-butyl, n-hexyl, n-heptyl, n-octyl,n-nonyl, n-decyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl,1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl,4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl,2,3-dimethyl-2-butyl, and 3,3-dimethyl-2-butyl.

Alkyl groups, whether alone or as part of another group, can beoptionally substituted with one or more groups, preferably 1 to 3 groups(and any additional substituents selected from halogen), including, butnot limited to, -halogen, —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl),—O—(C₂-C₈ alkynyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂,—C(O)NHR′, —C(O)N(R′)₂, —NHC(O)R′, —SR′, —SO₃R′, —S(O)₂R′, —S(O)R′, —OH,═O, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN, where each R′ is independentlyselected from —H, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or-aryl, and wherein said —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈alkynyl), -aryl, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, and —C₂-C₈ alkynyl groupscan be optionally further substituted with one or more groups including,but not limited to, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl,-halogen, —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈ alkynyl),-aryl, —C(O)R″, —OC(O)R″, —C(O)OR″, —C(O)NH₂, —C(O)NHR″, —C(O)N(R″)₂,—NHC(O)R″, —SR″, —SO₃R″, —S(O)₂R″, —S(O)R″, —OH, —N₃, —NH₂, —NH(R″),—N(R″)₂ and —CN, where each R″ is independently selected from —H, —C₁-C₈alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or -aryl.

Unless otherwise noted, the terms “alkenyl” and “alkynyl” refer tostraight and branched carbon chains having from about 2 to about 20carbon atoms (and all combinations and subcombinations of ranges andspecific numbers of carbon atoms therein), with from about 2 to about 8carbon atoms being preferred. An alkenyl chain has at least one doublebond in the chain and an alkynyl chain has at least one triple bond inthe chain. Examples of alkenyl groups include, but are not limited to,ethylene or vinyl, allyl, -1-butenyl, -2-butenyl, -isobutylenyl,-1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, and-2,3-dimethyl-2-butenyl. Examples of alkynyl groups include, but are notlimited to, acetylenic, propargyl, acetylenyl, propynyl, -1-butynyl,-2-butynyl, -1-pentynyl, -2-pentynyl, and -3-methyl-1 butynyl.

Alkenyl and alkynyl groups, whether alone or as part of another group,can be optionally substituted with one or more groups, preferably 1 to 3groups (and any additional substituents selected from halogen),including but not limited to, -halogen, —O—(C₁-C₈ alkyl), —O—(C₂-C₈alkenyl), —O—(C₂-C₈ alkynyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′,—C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂, —NHC(O)R′, —SR′, —SO₃R′, —S(O)₂R′,—S(O)R′, —OH, ═O, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN, where each R′ isindependently selected from —H, —C₁-C₈ alkyl, —C₂-C₈ alkyenl, —C₂-C₈alkynyl, or -aryl and wherein said —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl),—O—(C₂-C₈ alkynyl), -aryl, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, and —C₂-C₈alkynyl groups can be optionally further substituted with one or moresubstituents including, but not limited to, —C₁-C₈ alkyl, —C₂-C₈alkenyl, —C₂-C₈ alkynyl, -halogen, —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl),—O—(C₂C₈ alkynyl), -aryl, —C(O)R″, —OC(O)R″, —C(O)OR″, —C(O)NH₂,—C(O)NHR″, —C(O)N(R″)₂, —NHC(O)R″, —SR″, —SO₃R″, —S(O)₂R″, —S(O)R″, —OH,—N₃, —NH₂, —NH(R″), —N(R″)₂ and —CN, where each R″ is independentlyselected from —H, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or-aryl.

Unless otherwise noted, the term “alkylene” refers to a saturatedbranched or straight chain hydrocarbon radical having from about 1 toabout 20 carbon atoms (and all combinations and subcombinations ofranges and specific numbers of carbon atoms therein), with from about 1to about 8 carbon atoms being preferred and having two monovalentradical centers derived by the removal of two hydrogen atoms from thesame or two different carbon atoms of a parent alkane. Typical alkylenesinclude, but are not limited to, methylene, ethylene, propylene,butylene, pentylene, hexylene, heptylene, ocytylene, nonylene, decalene,1,4-cyclohexylene, and the like. Alkylene groups, whether alone or aspart of another group, can be optionally substituted with one or moregroups, preferably 1 to 3 groups (and any additional substituentsselected from halogen), including, but not limited to, -halogen,—O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈ alkynyl), -aryl,—C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂,—NHC(O)R′, —SR′, —SO₃R′, —S(O)₂R′, —S(O)R′, —OH, ═O, —N₃, —NH₂, —NH(R′),—N(R′)₂ and —CN, where each R′ is independently selected from —H, —C₁-C₈alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or -aryl and wherein said—O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈ alkynyl), -aryl, —C₁-C₈alkyl, —C₂-C₈ alkenyl, and —C₂-C₈ alkynyl groups can be furtheroptionally substituted with one or more substituents including, but notlimited to, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, -halogen,—O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈ alkynyl), -aryl,—C(O)R″, —OC(O)R″, —C(O)OR″, —C(O)NH₂, —C(O)NHR″, —C(O)N(R″)₂,—NHC(O)R″, —SR″, —SO₃R″, —S(O)₂R″, —S(O)R″, —OH, —N₃, —NH₂, —NH(R″),—N(R″)₂ and —CN, where each R″ is independently selected from —H, —C₁-C₈alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or -aryl.

Unless otherwise noted, the term “alkenylene” refers to an optionallysubstituted alkylene group containing at least one carbon-carbon doublebond. Exemplary alkenylene groups include, for example, ethenylene(—CH═CH—) and propenylene (—CH═CHCH₂—).

Unless otherwise noted, the term “alkynylene” refers to an optionallysubstituted alkylene group containing at least one carbon-carbon triplebond. Exemplary alkynylene groups include, for example, acetylene(—C≡C—), propargyl (—CH₂C≡C—), and 4-pentynyl (—CH₂CH₂CH₂C≡CH—).

Unless otherwise noted, the term “aryl” refers to a monovalent aromatichydrocarbon radical of 6-20 carbon atoms (and all combinations andsubcombinations of ranges and specific numbers of carbon atoms therein)derived by the removal of one hydrogen atom from a single carbon atom ofa parent aromatic ring system. Some aryl groups are represented in theexemplary structures as “Ar”. Typical aryl groups include, but are notlimited to, radicals derived from benzene, substituted benzene, phenyl,naphthalene, anthracene, biphenyl, and the like.

An aryl group, whether alone or as part of another group, can beoptionally substituted with one or more, preferably 1 to 5, or even 1 to2 groups including, but not limited to, -halogen, —C₁-C₈ alkyl, —C₂-C₈alkenyl, —C₂-C₈ alkynyl, —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈alkynyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′,—C(O)N(R′)₂, —NHC(O)R′, —SR′, —SO₃R′, —S(O)₂R′, —S(O)R′, —OH, —NO₂, —N₃,—NH₂, —NH(R′), —N(R′)₂ and —CN, where each R′ is independently selectedfrom —H, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or -aryl andwherein said —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, O—(C₁-C₈alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈ alkynyl), and -aryl groups can befurther optionally substituted with one or more substituents including,but not limited to, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl,-halogen, —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈ alkynyl),-aryl, —C(O)R″, —OC(O)R″, —C(O)OR″, —C(O)NH₂, —C(O)NHR″, —C(O)N(R″)₂,—NHC(O)R″, —SR″, —SO₃R″, —S(O)₂R″, —S(O)R″, —OH, —N₃, —NH₂, —NH(R″),—N(R″)₂ and —CN, where each R″ is independently selected from —H, —C₁-C₈alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or -aryl.

Unless otherwise noted, the term “arylene” refers to an optionallysubstituted aryl group which is divalent (i.e., derived by the removalof two hydrogen atoms from the same or two different carbon atoms of aparent aromatic ring system) and can be in the ortho, meta, or paraconfigurations as shown in the following structures with phenyl as theexemplary aryl group:

Typical “—(C₁-C₈ alkylene)aryl,” “—(C₂-C₈ alkenylene)aryl”, “and —(C₂-C₈alkynylene)aryl” groups include, but are not limited to, benzyl,2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl,2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl,2-naphthophenylethan-1-yl and the like.

Unless otherwise noted, the term “heterocycle,” refers to a monocyclic,bicyclic, or polycyclic ring system having from 3 to 14 ring atoms (alsoreferred to as ring members) wherein at least one ring atom in at leastone ring is a heteroatom selected from N, O, P, or S (and allcombinations and subcombinations of ranges and specific numbers ofcarbon atoms and heteroatoms therein). The heterocycle can have from 1to 4 ring heteroatoms independently selected from N, O, P, or S. One ormore N, C, or S atoms in a heterocycle can be oxidized. A monocylicheterocycle preferably has 3 to 7 ring members (e.g., 2 to 6 carbonatoms and 1 to 3 heteroatoms independently selected from N, O, P, or S),and a bicyclic heterocycle preferably has 5 to 10 ring members (e.g., 4to 9 carbon atoms and 1 to 3 heteroatoms independently selected from N,O, P, or S). The ring that includes the heteroatom can be aromatic ornon-aromatic. Unless otherwise noted, the heterocycle is attached to itspendant group at any heteroatom or carbon atom that results in a stablestructure.

Heterocycles are described in Paquette, “Principles of ModemHeterocyclic Chemistry” (W.A. Benjamin, New York, 1968), particularlyChapters 1, 3, 4, 6, 7, and 9; “The Chemistry of Heterocyclic Compounds,A series of Monographs” (John Wiley & Sons, New York, 1950 to present),in particular Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc.82:5566 (1960).

Unless otherwise noted, the term “heterocyclo” refers to an optionallysubstituted heterocycle group as defined above that is divalent (i.e.,derived by the removal of two hydrogen atoms from the same or twodifferent carbon atoms of a parent heterocyclic ring system).

Examples of “heterocycle” groups include by way of example and notlimitation pyridyl, dihydropyridyl, tetrahydropyridyl (piperidyl),thiazolyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl,imidazolyl, tetrazolyl, benzofuranyl, thianaphthalenyl, indolyl,indolenyl, quinolinyl, isoquinolinyl, benzimidazolyl, piperidinyl,4-piperidonyl, pyrrolidinyl, 2-pyrrolidonyl, pyrrolinyl,tetrahydrofuranyl, bis-tetrahydrofuranyl, tetrahydropyranyl,bis-tetrahydropyranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,decahydroquinolinyl, octahydroisoquinolinyl, azocinyl, triazinyl,6H-1,2,5-thiadiazinyl, 2H,6H-1,5,2-dithiazinyl, thienyl, thianthrenyl,pyranyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxathinyl,2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl,indolizinyl, isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl,4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl,quinazolinyl, cinnolinyl, pteridinyl, 4H-carbazolyl, carbazolyl,β-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl,chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl,piperazinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl,oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl,and isatinoyl. Preferred “heterocycle” groups include, but are notlimited to, benzofuranyl, benzothiophenyl, indolyl, benzopyrazolyl,coumarinyl, isoquinolinyl, pyrrolyl, thiophenyl, furanyl, thiazolyl,imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl, pyridinyl,pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl andtetrazolyl.

A heterocycle group, whether alone or as part of another group, can beoptionally substituted with one or more groups, preferably 1 to 2groups, including but not limited to, —C₁-C₈ alkyl, —C₂-C₈ alkenyl,—C₂-C₈ alkynyl, -halogen, —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl),—O—(C₂-C₈ alkynyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂,—C(O)NHR′, —C(O)N(R′)₂, —NHC(O)R′, —SR′, —SO₃R′, —S(O)₂R′, —S(O)R′, —OH,—N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN, where each R′ is independentlyselected from —H, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or -aryland wherein said —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈alkynyl), —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, and -aryl groupscan be further optionally substituted with one or more substituentsincluding, but not limited to, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈alkynyl, -halogen, —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈alkynyl), -aryl, —C(O)R″, —OC(O)R″, —C(O)OR″, —C(O)NH₂, —C(O)NHR″,—C(O)N(R″)₂, —NHC(O)R″, —SR″, —SO₃R″, —S(O)₂R″, —S(O)R″, —OH, —N₃, —NH₂,—NH(R″), —N(R″)₂ and —CN, where each R″ is independently selected from—H, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or aryl.

Unless otherwise noted, the term “carbocycle,” refers to a saturated orunsaturated non-aromatic monocyclic, bicyclic, or polycyclic ring systemhaving from 3 to 14 ring atoms (and all combinations and subcombinationsof ranges and specific numbers of carbon atoms therein) wherein all ofthe ring atoms are carbon atoms. Monocyclic carbocycles preferably have3 to 6 ring atoms, still more preferably 5 or 6 ring atoms. Bicycliccarbocycles preferably have 7 to 12 ring atoms, e.g., arranged as abicyclo[4,5], [5,5], [5,6] or [6,6] system, or 9 or 10 ring atomsarranged as a bicyclo[5,6] or [6,6] system. The term “carbocycle”includes, for example, a monocyclic carbocycle ring fused to an arylring (e.g., a monocyclic carbocycle ring fused to a benzene ring).Carbocyles preferably have 3 to 8 carbon ring atoms.

Carbocycle groups, whether alone or as part of another group, can beoptionally substituted with, for example, one or more groups, preferably1 or 2 groups (and any additional substituents selected from halogen),including, but not limited to, -halogen, —C₁-C₈ alkyl, —C₂-C₈ alkenyl,—C₂-C₈ alkynyl, —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈alkynyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′,—C(O)N(R′)₂, —NHC(O)R′, —SR′, —SO₃R′, —S(O)₂R′, —S(O)R′, —OH, ═O, —N₃,—NH₂, —NH(R′), —N(R′)₂ and —CN, where each R′ is independently selectedfrom —H, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or -aryl andwherein said —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, —O—(C₁-C₈alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈ alkynyl), and -aryl groups can befurther optionally substituted with one or more substituents including,but not limited to, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl,-halogen, —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈ alkynyl),-aryl, —C(O)R″, —OC(O)R″, —C(O)OR″, —C(O)NH₂, —C(O)NHR″, —C(O)N(R″)₂,—NHC(O)R″, —SR″, —SO₃R″, —S(O)₂R″, —S(O)R″, —OH, —N₃, —NH₂, —NH(R″),—N(R″)₂ and —CN, where each R″ is independently selected from —H, —C₁-C₈alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or -aryl.

Examples of monocyclic carbocylic substituents include -cyclopropyl,-cyclobutyl, -cyclopentyl, -1-cyclopent-1-enyl, -1-cyclopent-2-enyl,-1-cyclopent-3-enyl, cyclohexyl, -1-cyclohex-1-enyl, -1-cyclohex-2-enyl,-1-cyclohex-3-enyl, -cycloheptyl, -cyclooctyl.-1,3-cyclohexadienyl,-1,4-cyclohexadienyl, -1,3-cycloheptadienyl, -1,3,5-cycloheptatrienyl,and -cyclooctadienyl.

A “carbocyclo,” whether used alone or as part of another group, refersto an optionally substituted carbocycle group as defined above that isdivalent (i.e., derived by the removal of two hydrogen atoms from thesame or two different carbon atoms of a parent carbocyclic ring system).

Unless otherwise indicated by context, a hyphen (-) designates the pointof attachment to the pendant molecule. Accordingly, the term “—(C₁-C₈alkylene)aryl” or “—C₁-C₈ alkylene(aryl)” refers to a C₁-C₈ alkyleneradical as defined herein wherein the alkylene radical is attached tothe pendant molecule at any of the carbon atoms of the alkylene radicaland one of the hydrogen atoms bonded to a carbon atom of the alkyleneradical is replaced with an aryl radical as defined herein.

When a particular group is “substituted”, that group may have one ormore substituents, preferably from one to five substituents, morepreferably from one to three substituents, most preferably from one totwo substituents, independently selected from the list of substituents.Groups that are substituted are so indicated.

It is intended that the definition of any substituent or variable at aparticular location in a molecule be independent of its definitionselsewhere in that molecule. It is understood that substituents andsubstitution patterns on the compounds of this invention can be selectedby one of ordinary skill in the art to provide compounds that arechemically stable and that can be readily synthesized by techniquesknown in the art as well as those methods set forth herein.

C. Anti-CD30 Antibodies

Anti-CD30 antibodies suitable for use in accordance with the presentcompositions and methods include any antibody that specifically binds tothe CD30 antigen.

Anti-CD30 antibodies of the present invention are preferably monoclonaland can include, for example, chimeric (e.g., having a human constantregion and mouse variable region), humanized, or human antibodies.Preferably, the immunoglobulin molecule is of the IgG type and can beany subclass (e.g., IgG1, IgG2, IgG3, IgG4) of immunoglobulin moleculeand variants thereof. The immunoglobulin molecule is most preferably anIgG1.

The antibodies of the present invention can be generated by any suitablemethod known in the art. Monoclonal antibodies can be prepared using awide variety of techniques known in the art including the use ofhybridoma, recombinant, and phage display technologies, or a combinationthereof. For example, monoclonal antibodies can be produced usinghybridoma techniques including those known in the art and taught, forexample, in Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2nd ed., 1988); Hammerling, et al., in:Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y.,1981) (said references incorporated by reference in their entireties).

Any method known in the art for the synthesis of proteins, e.g.,recombinant expression techniques, can be used to generate the anti-CD30antibodies of the present invention.

Once a CD30-binding protein is identified, if desired, its ability(alone or when multimerized or fused to a dimerization ormultimerization domain) to elicit a cytostatic or cytotoxic effect onCD30-expressing cancerous cells can be determined by contacting aculture of an CD30-expressing cancer cell line, such as L428, L450,HLLM2 or KM-H2, with the protein. Culture conditions are most preferablyabout 5,000 cells in a culture area of about 0.33 cm², and thecontacting period being approximately 72 hours. The culture is thenexposed to 0.5 μCi of ³H-thymidine during the final 8 hours of the72-hour period and the incorporation of ³H-thymidine into cells of theculture is measured. The protein has a cytostatic or cytotoxic effect onthe cell line if the cells of the culture have reduced ³H-thymidineincorporation compared to cells of the same cell line cultured under thesame conditions but not contacted with the protein. There are many othercytotoxicity assays known to those of skill in the art. Any one of themcan be used in the present methods.

Exemplary anti-CD30 antibodies include, but are not limited to,humanized or chimeric AC10 or HeFi-1 antibodies. Murine AC10 has beendeposited under ATCC Accession Number PTA-6679.

An exemplary anti-CD30 antibody comprises one or more (1, 2, 3, 4, 5, or6) CDRs of murine HeFi-1 (SEQ ID NO:20, SEQ ID NO:22; SEQ ID NO:24; SEQID NO:28, SEQ ID NO:30 or SEQ ID NO:32) or murine AC10 (SEQ ID NO:4; SEQID NO:6; SEQ ID NO:8; SEQ ID NO:12; SEQ ID NO:14; or SEQ ID NO:16). Insome embodiments, the anti-CD30 antibody comprises the heavy and/orlight chain variable regions of murine HeFi-1 (SEQ ID NO:18 or SEQ IDNO:26) or murine AC10 (SEQ ID NO:2 or SEQ ID NO:10). A table indicatingthe region of AC10 or HeFi-1 to which each SEQ ID NO corresponds to isprovided below.

TABLE 1 NUCLEOTIDE OR MOLECULE AMINO ACID SEQ ID NO AC10 Heavy ChainVariable Region Nucleotide 1 AC10 Heavy Chain Variable Region Amino Acid2 AC10 Heavy Chain-CDR1(H1) Nucleotide 3 AC10 Heavy Chain-CDR1(H1) AminoAcid 4 AC10 Heavy Chain-CDR2(H2) Nucleotide 5 AC10 Heavy Chain-CDR2(H2)Amino Acid 6 AC10 Heavy Chain-CDR3(H3) Nucleotide 7 AC10 HeavyChain-CDR3(H3) Amino Acid 8 AC10 Light Chain Variable Region Nucleotide9 AC10 Light Chain Variable Region Amino Acid 10 AC10 LightChain-CDR1(L1) Nucleotide 11 AC10 Light Chain-CDR1(L1) Amino Acid 12AC10 Light Chain-CDR2(L2) Nucleotide 13 AC10 Light Chain-CDR2(L2) AminoAcid 14 AC10 Light Chain-CDR3(L3) Nucleotide 15 AC10 LightChain-CDR3(L3) Amino Acid 16 HeFi-1 Heavy Chain Variable RegionNucleotide 17 HeFi-1 Heavy Chain Variable Region Amino Acid 18 HeFi-1Heavy Chain-CDR1(H1) Nucleotide 19 HeFi-1 Heavy Chain-CDR1(H1) AminoAcid 20 HeFi-1 Heavy Chain-CDR2(H2) Nucleotide 21 HeFi-1 HeavyChain-CDR2(H2) Amino Acid 22 HeFi-1 Heavy Chain-CDR3(H3) Nucleotide 23HeFi-1 Heavy Chain-CDR3(H3) Amino Acid 24 HeFi-1 Light Chain VariableRegion Nucleotide 25 HeFi-1 Light Chain Variable Region Amino Acid 26HeFi-1 Light Chain-CDR1(L1) Nucleotide 27 HeFi-1 Light Chain-CDR1(L1)Amino Acid 28 HeFi-1 Light Chain-CDR2(L2) Nucleotide 29 HeFi-1 LightChain-CDR2(L2) Amino Acid 30 HeFi-1 Light Chain-CDR3(L3) Nucleotide 31HeFi-1 Light Chain-CDR3(L3) Amino Acid 32 Human gamma I constant regionAmino Acid 33 Human kappa constant region Amino Acid 34

In a specific embodiment, the invention encompasses an antibodycomprising a heavy chain variable domain, said variable domaincomprising (a) a set of three CDRs, in which said set of CDRs comprisesSEQ ID NO:4, 6, or 8 or comprises amino acid sequences that aresubstantially identical to the amino acid sequences set forth in SEQ IDNO:4, 6, or 8 and (b) a set of four framework regions, in which said setof framework regions differs from the set of framework regions in murinemonoclonal antibody AC10, and in which said antibody immunospecificallybinds CD30.

In a specific embodiment, the invention encompasses an antibodycomprising a heavy chain variable domain, said variable domaincomprising (a) a set of three CDRs, in which said set of CDRs comprisesSEQ ID NO:20, 22 or 24 or comprises amino acid sequences that aresubstantially identical to the amino acid sequences set forth in SEQ IDNO:20, 22, or 24 and (b) a set of four framework regions, in which saidset of framework regions differs from the set of framework regions inmurine monoclonal antibody HeFi-1, and in which said antibodyimmunospecifically binds CD30.

In a specific embodiment, the invention encompasses an antibodycomprising a light chain variable domain, said variable domaincomprising (a) a set of three CDRs, in which said set of CDRs comprisesSEQ ID NO:12, 14 or 16 or comprises amino acid sequences that aresubstantially identical to the amino acid sequences set forth in SEQ IDNO:12, 14, or 16, and (b) a set of four framework regions, in which saidset of framework regions differs from the set of framework regions inmurine monoclonal antibody AC10, and in which said antibodyimmunospecifically binds CD30.

In a specific embodiment, the invention encompasses an antibodycomprising a light chain variable domain, said variable domaincomprising (a) a set of three CDRs, in which said set of CDRs comprisesSEQ ID NO:28, 30, or 32 or comprises amino acid sequences that aresubstantially identical to the amino acid sequences set forth in SEQ IDNO:28, 30, or 32, and (b) a set of four framework regions, in which saidset of framework regions differs from the set of framework regions inmurine monoclonal antibody HeFi-1, and in which said antibodyimmunospecifically binds CD30.

In certain embodiments, the anti-CD30 antibody comprises a gamma Iconstant region, (e.g., huCγ1, SwissProt accession number P01857) and ahuman kappa constant region (e.g., huCκ, PID G185945).

The present invention encompasses embodiments wherein a chimeric AC10antibody comprises the heavy chain variable region set forth in SEQ IDNO:2, the light chain variable region set forth in SEQ ID NO:10, thehuman gamma I constant region set forth in SEQ ID NO:33 (or amino acids1 to 329 of SEQ ID NO:33) and the human kappa constant region set forthin SEQ ID NO:34.

Additionally, the antibodies can also be described or specified in termsof their primary structures. In some embodiments, the variable regionsof the anti-CD30 antibodies will have at least 80%, at least 85%, atleast 90%, at least 95% and most preferably at least 98% identity (ascalculated using methods known in the art and described herein) to thevariable regions of murine AC10 or HeFi-1.

Exemplary anti-CD30 antibodies include functional derivatives or analogsof AC10 and HeFi-1. As used herein, the term “functional” in thiscontext indicates that the functional derivate or analog of AC10 andHeFi-1 is capable of binding to CD3O.

Antibodies for use in the present invention include those thatcompetitively inhibit binding of murine AC10 or HeFi-1 to CD30 asdetermined by any method known in the art for determining competitivebinding. For example, the antibody can inhibit binding of AC10 or HeFi-1to CD30 by at least 50%, at least 60%, at least 70%, at least 80%, atleast 85%, at least 90%, or even at least 95%.

D. Anti-CD30 vc-PAB-MMAE Antibody-Drug Conjugates

The methods described herein encompass the use of antibody drugconjugates comprising an anti-CD30 antibody, covalently linked to MMAEthrough a vc-PAB linker to treat a subject that has relapsed followingtreatment with a CD30 targeted therapy. After administration of theantibody-drug conjugate to a subject and binding of the anti-CD30antibody to a CD30 expressing cancer cell, the antibody drug conjugateinternalizes into the cell, and the drug is released.

The anti-CD30 vc-PAB-MMAE antibody-drug conjugates described herein haveFormula I:

or a pharmaceutically acceptable salt thereof;

wherein:

mAb is the antibody,

S is a sulfur atom of the antibody

A- is a Stretcher unit,

p is from about 2 to about 8.

The drug loading is represented by p, the average number of drugmolecules per antibody in a pharmaceutical composition. For example, ifp is about 4, the average drug loading taking into account all of theantibody present in the pharmaceutical composition is about 4. P rangesfrom about 2 to about 8, about 2 to about 5, about 3 to about 5, morepreferably from 3.6 to 4.4, even more preferably from 3.8 to 4.2. P canbe about 2, about 3, about 4, about 5, about 6, about 7 or about 8. Theaverage number of drugs per antibody in preparation of conjugationreactions may be characterized by conventional means such as massspectroscopy, ELISA assay, and HPLC. The quantitative distribution ofantibody-drug conjugates in terms of p may also be determined. In someinstances, separation, purification, and characterization of homogeneousantibody-drug-conjugates where p is a certain value fromantibody-drug-conjugates with other drug loadings may be achieved bymeans such as reverse phase HPLC or electrophoresis.

The Stretcher unit (A), is capable of linking an antibody to thevaline-citrulline amino acid unit via a sulfhydryl group of theantibody. Sulfhydryl groups can be generated, for example, by reductionof the interchain disulfide bonds of an anti-CD30 antibody. For example,the Stretcher unit can be linked to the antibody via the sulfur atomsgenerated from reduction of the interchain disulfide bonds of theantibody. In some embodiments, the Stretcher units are linked to theantibody solely via the sulfur atoms generated from reduction of theinterchain disulfide bonds of the antibody. In some embodiments, therecombinant anti-CD30 antibody is engineered to carry additionalsulfhydryl groups, e.g., cysteines are introduced into the antibody. Insome of these embodiments, the Stretcher units are linked to theantibody solely via sulfur atoms of the introduced cysteine residues. Insome embodiments, sulfhydryl groups are generated by reaction of anamino group of a lysine moiety of an anti-CD30 antibody with2-iminothiolane (Traut's reagent) or other sulfhydryl generatingreagents. In certain embodiments, the anti-CD30 antibody is arecombinant antibody and is engineered to carry one or more lysines.

The synthesis and structure of MMAE is described in U.S. Pat. No.6,884,869 incorporated by reference herein in its entirety and for allpurposes. The synthesis and structure of exemplary stretcher units andmethods for making antibody drug conjugates are described in, forexample, U.S. Publication Nos. 2006/0074008 and 2009/0010945 each ofwhich is incorporated herein by reference in its entirety.

Representative Stretcher units are depicted within the square bracketsof Formulas IVa and IVb and R₁₇ is selected from —C₁-C₁₀ alkylene-,—C₁-C₁₀ alkenylene-, —C₁-C₁₀ alkynylene-, -carbocyclo-, —O—(C₁-C₈alkylene)-, O—(C₁-C₈ alkenylene)-, —O—(C₁-C₈ alkynylene)-, -arylene-,—C₁-C₁₀ alkylene-arylene-, —C₂-C₁₀ alkenylene-arylene, —C₂-C₁₀alkynylene-arylene, -arylene-C₁-C₁₀ alkylene-, -arylene-C₂-C₁₀alkenylene-, -arylene-C₂-C₁₀ alkynylene-, —C₁-C₁₀alkylene-(carbocyclo)-, —C₂-C₁₀ alkenylene-(carbocyclo)-, —C₂-C₁₀alkynylene-(carbocyclo)-, -(carbocyclo)-C₁-C₁₀ alkylene-,-(carbocyclo)-C₂-C₁₀ alkenylene-, -(carbocyclo)-C₂-C₁₀ alkynylene,heterocyclo-, —C₁-C₁₀ alkylene-(heterocyclo)-, —C₂-C₁₀alkenylene-(heterocyclo)-, —C₂-C₁₀ alkynylene-(heterocyclo)-,-(heterocyclo)-C₁-C₁₀ alkylene-, -(heterocyclo)-C₂-C₁₀ alkenylene-,-(heterocyclo)-C₁-C₁₀ alkynylene-, —(CH₂CH₂O)_(r)—, or—(CH₂CH₂O)_(r)—CH₂—, and r is an integer ranging from 1-10, wherein saidalkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynyklene, aryl,carbocyle, carbocyclo, heterocyclo, and arylene radicals, whether aloneor as part of another group, are optionally substituted.

An illustrative Stretcher unit is that of Formula IVa wherein R¹⁷ is—(CH₂)₅—:

Another illustrative Stretcher unit is that of Formula IVa wherein R¹⁷is —(CH₂CH₂O)_(r)—CH₂—; and r is 2:

An illustrative Stretcher unit is that of Formula IVa wherein R¹⁷ is-arylene- or arylene-C₁-C₁₀ alkylene-. In some embodiments, the arylgroup is a phenyl group.

Still another illustrative Stretcher unit is that of Formula IVb whereinR¹⁷ is —(CH₂)₅—:

One way that the Stretcher unit can be linked to the antibody unit isvia a disulfide bond between a sulfur atom of the antibody unit and asulfur atom of the Stretcher unit. In some embodiments, the sulfur atomis from an internal cysteine residue of the antibody. In some otherembodiments, the sulfur atom is from a cysteine residue that has beenengineered into the antibody.

A representative Stretcher unit is depicted within the square bracketsof Formula V, wherein R¹⁷ is as defined above.

It should be noted that throughout this application, the S moiety in theformula below refers to a sulfur atom of the antibody, unless otherwiseindicated by context.

In a particularly preferred embodiment, of the present invention, thestrectcher is of Formula IVa and the antibody drug conjugate is aMC-vc-PAB-MMAE antibody drug conjugate as follows wherein p is fromabout 3 to about 5, preferably from 3.8 to 4.2. The S moiety refers to asulfur atom of the antibody.

In a particularly preferred embodiment, the antibody is a chimeric orhumanized AC10 antibody. In a particularly preferred embodiment, the Smoiety is generated by reduction of the interchain disulfide bonds ofthe antibody.

E. Pharmaceutical Compositions and Formulations

Various delivery systems can be used to administer the anti-CD30vc-PAB-MMAE antibody-drug conjugates. In certain preferred embodimentsof the present invention, administration of the antibody-drug conjugatecompound is by intravenous infusion. In some embodiments, administrationis by a two hour intravenous infusion.

The anti-CD30 vc-PAB-MMAE antibody-drug conjugates can be administeredas a pharmaceutical composition comprising one or more pharmaceuticallycompatible ingredients. For example, the pharmaceutical compositiontypically includes one or more pharmaceutically acceptable carriers, forexample, water-based carriers (e.g., sterile liquids). Water is a moretypical carrier when the pharmaceutical composition is administeredintravenously.

The composition, if desired, can also contain, for example, salinesalts, buffers, salts, nonionic detergents, and/or sugars. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. The formulations correspond tothe mode of administration.

The present invention provides, for example, pharmaceutical compositionscomprising a therapeutically effective amount of anti-CD30 vc-PAB-MMAEantibody-drug conjugates, a buffering agent, optionally acryoprotectant, optionally a bulking agent, optionally a salt, andoptionally a surfactant. Additional agents can be added to thecomposition. A single agent can serve multiple functions. For example, asugar, such as trehalose, can act as both a cryoprotectant and a bulkingagent. Any suitable pharmaceutically acceptable buffering agents,surfactants, cyroprotectants and bulking agents can be used inaccordance with the present invention.

In some embodiments, the anti-CD30 vc-PAB-MMAE antibody-drug conjugatesformulation comprises (i) about 1-25 mg/ml, preferably about 3 to about10 mg/ml of antibody-drug conjugates (e.g., antibody-drug conjugates offormula I or a pharmaceutically acceptable salt thereof), (ii) about5-50 mM, preferably about 10 mM to about 25 mM of a buffer selected froma citrate, phosphate, or histidine buffer or combinations thereof,preferably sodium citrate, potassium phosphate, histidine, histidinehydrochloride, or combinations thereof, (iii) about 3% to about 10%sucrose or trehalose or combinations thereof, (iv) optionally about 0.05to 2 mg/ml of a surfactant selected from polysorbate 20 or polysorbate80 or combinations thereof; and (v) water, wherein the pH of thecomposition is from about 5.3 to about 7, preferably about 6.6.

The antibody drug conjugate formulation can, for example, comprise about5 mg/ml of an antibody-drug conjugate, (ii) about 20 mM sodium citrate(iii) about 6% to about 7% trehalose (about 70 mg/ml), (iv) about 0.1 to0.3 mg/ml of a surfactant selected from polysorbate 20 or polysorbate80, and (v) water, wherein the pH of the composition is from about 5.3to about 7, preferably about 6.6.

The amount of the anti-CD30 vc-PAB-MMAE antibody-drug conjugates to beadministered can be determined by standard clinical techniques. Theprecise dose to be employed in the compositions will also depend on theroute of administration, and the seriousness of the disease or disorder,and should be decided according to the judgment of the practitioner andeach patient's circumstances. In some embodiments, the anti-CD30vc-PAB-MMAE antibody-drug conjugates will be administered at a dosage offrom 1 mg/kg to 2.4 mg/kg every three weeks for at least 6 weeks oftreatment, preferably a dosage of from 1.6 mg/kg to 2 mg/kg every threeweeks for at least 6 weeks of treatment, and most preferably a dosage ofabout 1.8 mg/kg every three weeks for at least 6 weeks of treatment. Insome embodiments, the anti-CD30 vc-PAB-MMAE antibody-drug conjugateswill be administered at a dosage of from 0.6 mg/kg to 1.4 mg/kg, morepreferably from 0.8 mg/kg to 1.2 mg/kg and most preferably at a dosageof about 1 mg/kg weekly 3 out of 4 weeks (e.g., on days 1, 8, and 15 ofa 28 day treatment cycle) for 1, 2, 3, 4, 5, or 6 four week treatmentcycles.

In some embodiments, wherein the subject had previously been treatedwith an anti-CD30 vc-PAB-MMAE antibody-drug conjugate, administrationwas at a dosage of from 1 mg/kg to 2.4 mg/kg every three weeks for atleast 6 weeks of treatment, preferably at a dosage of from 1.6 mg/kg to2 mg/kg every three weeks for at least 6 weeks of treatment, and mostpreferably at a dosage of about 1.8 mg/kg every three weeks for at least6 weeks of treatment. In some embodiments, administration was at adosage of from 0.6 mg/kg to 1.4 mg/kg, more preferably from 0.8 mg/kg to1.2 mg/kg and most preferably at a dosage of about 1 mg/kg weekly 3 outof 4 weeks (e.g., on days 1, 8, and 15 of a 28 day treatment cycle) for1, 2, 3, 4, 5, or 6 four week treatment cycles.

The present invention is not to be limited in scope by the specificembodiments described herein. Various modifications of the invention inaddition to those described herein will become apparent to those skilledin the art from the foregoing description and accompanying figures. Suchmodifications are intended to fall within the scope of the appendedclaims.

All publications and patent documents cited above are herebyincorporated by reference in their entirety for all purposes to the sameextent as if each were so individually denoted.

The invention is further described in the following examples, which arein not intended to limit the scope of the invention.

EXAMPLES

Background:

Brentuximab vedotin is an anti-CD30 antibody conjugated to the highlypotent antitubulin agent, monomethyl auristatin E (MMAE), by aplasma-stable linker. Brentuximab vedotin selectively induces apoptosisin CD30+ cells by binding, internalizing, and releasing MMAE. In phase 1clinical studies, antitumor activity was demonstrated in patients withCD30+ hematologic malignancies. This case series describes patients whohave experienced relapse and were retreated with brentuximab vedotin.

Methods:

Three multicenter studies have enrolled heavily pretreated, relapsed orrefractory patients with CD30+ hematologic malignancies who experiencedclinical benefit with prior brentuximab vedotin treatment (stabledisease with decreasing tumor volume or better; Cheson 2007), andsubsequently relapsed. In their retreatment experiences, patientsreceived brentuximab vedotin IV infusions of 1 mg/kg weekly or 1.8 mg/kgevery 3 weeks.

Results:

8 pateints received retreatment with brentuximab vedotin; 3 patientswere retreated twice. Patients had either ALCL or HL; ages ranged from16-39 years. At baseline prior to retreatment, patients had an ECOGperformance status of 0/1 or 2. All treatment-related adverse eventswere G1/2 in severity. Among the retreatment experiences, there were 2complete responses, and 5 partial responses. Tumor regression wasobserved in 10 out of 11 retreatment patients. Most objective responseswere observed 5-15 weeks after the start of retreatment, and responsedurations of 58+ weeks have been demonstrated.

What is claimed is:
 1. A method for the post-relapse treatment of asubject having a previously treated CD30 expressing lymphoma comprisingadministering to the subject a pharmaceutical composition comprisingantibody-drug conjugates having formula I:

or a pharmaceutically acceptable salt thereof; and a pharmaceuticallyacceptable carrier; wherein: Ab is an anti-CD30 antibody, S is a sulfuratom of the antibody A- is a Stretcher unit, and p is from about 2 toabout 8; wherein the subject had previously been treated with CD30targeted therapy, had discontinued treatment with the CD30 targetedtherapy after achieving clinical benefit, and had subsequently relapsed,and wherein following the post-relapse treatment with the pharmaceuticalcomposition comprising antibody-drug conjugates having formula I, thesubject experiences tumor regression.
 2. The method of claim 1 whereinthe CD30 expressing lymphoma is Hodgkin lymphoma.
 3. The method of claim1 wherein the CD30 expressing lymphoma is anaplastic large celllymphoma.
 4. The method of claim 1 wherein the antibody-drug conjugatesare of the formula

or a pharmaceutically acceptable salt thereof.
 5. The method of claim 1wherein S is a sulfur atom of a cysteine residue of the antibody.
 6. Themethod of claim 4 wherein S is a sulfur atom of a cysteine residue ofthe antibody.
 7. The method of claim 1 wherein the anti-CD30 antibodycompetes for binding with a murine AC10 antibody.
 8. The method of claim4 wherein p is about
 4. 9. The method of claim 5 wherein p is about 4.10. The method of claim 6 wherein p is about
 4. 11. The method of claim7 wherein the anti-CD30 antibody is a chimeric AC10 antibody.
 12. Themethod of claim 11 wherein the CD30 targeted therapy is therapy with apharmaceutical composition comprising antibody-drug conjugates havingformula I or a pharmaceutically acceptable salt thereof.
 13. The methodof claim 1 wherein the subject has a complete response following postrelapse treatment with the pharmaceutical composition comprisingantibody-drug conjugates having formula I.
 14. The method of claim 1wherein the subject has a partial response following post relapsetreatment with the pharmaceutical composition comprising antibody-drugconjugates having formula I.
 15. The method of claim 1 wherein thepharmaceutical composition comprising antibody-drug conjugates havingFormula I is administered at a dosage of from 1 mg/kg to 2.4 mg/kg everythree weeks for at least 6 weeks of treatment or at a dosage of from 0.6mg/kg to 1.4 mg/kg, weekly 3 out of 4 weeks for 1, 2, 3, 4, 5, or 6 fourweek treatment cycles.
 16. A method for the post-relapse treatment of asubject having a previously treated CD30 expressing lymphoma comprisingadministering to the subject a pharmaceutical composition comprisingantibody-drug conjugates having formula I:

or a pharmaceutically acceptable salt thereof; and a pharmaceuticallyacceptable carrier; wherein: Ab is an anti-CD30 antibody, S is a sulfuratom of the antibody A- is a Stretcher unit, and p is from about 2 toabout 8; wherein the subject had previously been treated with apharmaceutical composition comprising antibody-drug conjugates havingformula I, had discontinued treatment after achieving clinical benefit,and had subsequently relapsed, and wherein following the post-relapseretreatment with the pharmaceutical composition comprising antibody-drugconjugates having formula I, the subject experiences tumor regression.17. The method of claim 16 wherein the pharmaceutical compositioncomprising antibody-drug conjugates having Formula I is administered ata dosage of from 1 mg/kg to 2.4 mg/kg every three weeks for at least 6weeks of treatment or at a dosage of from 0.6 mg/kg to 1.4 mg/kg, weekly3 out of 4 weeks for 1, 2, 3, 4, 5, or 6 four week treatment cycles.